Microtubule doublets (MTDs), consisting of an incomplete B-microtubule at the surface of a complete A-microtubule, provide a structural scaffold mediating intraflagellar transport and ciliary beating. Despite the fundamental role of MTDs, the molecular mechanism governing their formation is unknown. We used a cell-free assay to demonstrate a crucial inhibitory role of the carboxyl-terminal (C-terminal) tail of tubulin in MTD assembly. Removal of the C-terminal tail of an assembled A-microtubule allowed for the nucleation of a B-microtubule on its surface. C-terminal tails of only one A-microtubule protofilament inhibited this side-to-surface tubulin interaction, which would be overcome in vivo with binding protein partners. The dynamics of B-microtubule nucleation and its distinctive isotropic elongation was elucidated by using live imaging. Thus, inherent interaction properties of tubulin provide a structural basis driving flagellar MTD assembly.